植物内生菌定殖检测技术及其应用

自然环境中内生菌定殖于植物体内,对宿主植物产生多种有益效应,但是内生菌定殖情况难以检测,相关研究不够深入系统。目前在该领域使用较为广泛的检测技术包括：荧光标记、抗生素标记、荧光定量PCR和高通量测序等。内生菌通过孔隙伤口和降解细胞壁等方式侵染植物,通过种子垂直传递核心内生菌。对植物内生菌定殖的侵染方式、定殖方法和检测技术进行了归纳和整理,介绍了内生菌多种侵染和迁移途径,总结了目前内生菌定殖在生物防治、促进植物生长和污染修复等方面的功能,综述了多种检测方式在应用中的特点,以期为内生菌定殖植物的相关研究及其应用提供参考依据。     

植物内生细菌的分离、分类、定殖与应用

植物内生细菌已成为国内外的研究热点,目前已从植物中分离得到许多不同种属的植物内生细菌,其中有的具有促生与防治病害等对宿主植物有利的作用,而一些则具有潜在的致病性。研究人员利用多种方法对植物内生细菌的内生定殖行为进行研究,发现了有意义的定殖规律,例如,某种植物内生细菌对其宿主的侵染能力明显强于另一种内生细菌。目前,尽管植物内生细菌还有不少问题有待解决,但它在农业上的巨大应用潜力业已彰显。     

植物根际促生细菌定殖研究进展

植物促生菌(plant grwth-promoting bacterium,PGPB)因可有效抑制根际病原菌,促进植物生长,增加作物产量等作用,在农业生产领域展现出巨大前景。以了解植物根际促生细菌的种类,理解定殖相关的促生机制为目的,展示了根际细菌的定殖过程及影响细菌在根部定殖的因素,通过抗生素标记、免疫学方法、外源基因标记等方法进行定殖微生物的检测,以高通量测序技术在根际定殖中的广泛应用为结果,得出结论应从遗传水平上对菌株进行解读,获得植物根际土壤微生物群落多样性,功能基因等研究,这项工作对预测根际促生细菌与植物的交互作用,生物菌剂的田间应用有重要意义,应是植物根际促生细菌今后的研究方向。     

内生细菌在荔枝体内的定殖及其防病保鲜功能

用含有GFP基因标记的内生细菌菌株BS-2-gfp和TB2-gfp,采用喷雾接种的方法,研究其在荔枝叶片、花、幼果及成熟果实果皮内的定殖动态及其与防病保鲜之间的关系.结果表明:内生细菌BS-2-gfp和TB2-gfp能在荔枝叶片、花、幼果及采后果皮上定殖,能在各组织内繁殖并可在花和幼果间传导.内生细菌在荔枝叶片上的定殖因季节和荔枝生长期的不同而异,与秋季相比,春季定殖期限长,定殖量大.内生细菌在荔枝不同部位的定殖时间和定殖量也不同,在叶片上接种37d后还可分离回收到接种的2种目标细菌,在花上接种10d后就回收不到BS-2-gfp,而成熟荔枝果皮上2种菌的定殖量最大.防病试验表明,当荔枝霜疫病的病情指数急剧上升时,TB2-gfp在荔枝果皮的定殖量达到最大,为1.90×106CFU.g-1FM;保鲜试验发现,TB2-gfp菌株的保鲜效果优于BS-2-gfp,菌株在荔枝果皮内的定殖量也高于菌株BS-2-gfp,表明供试内生细菌在荔枝果皮的定殖量与其防病及保鲜效果呈正相关.     

植物内生酵母研究简况

当前国内、外对植物内生菌的研究主要集中在内生真菌、内生细菌的多样性及其相关应用等方面,而关于植物内生酵母多样性及应用的研究相对较少.目前研究表明,所分离出的植物内生酵母最常见于隐球菌属、德巴利酵母属、孢子酵母属和红酵母属,并且内生酵母在植物中定殖的具体机制尚未确定.概述了现有研究中关于植物内生酵母的研究现状及应用研究进...     

内生细菌01-144在番茄根茎内定殖的初步研究

对内生细菌 0 1 1 4 4进行了抗药性标记 ,利用标记菌株研究了其在番茄根茎内的定殖情况。浸种与灌根处理均可使 0 1 1 4 4在番茄根茎内定殖 ,且在根内的定殖能力大于茎内 ;灌根处理还表明 ,其在茎下部的定殖能力大于茎上部 ;0 1 1 4 4定殖数量动态在根茎内均有一个“由增到减”的趋势 ,但其在根内的数量变化明显较茎内平缓。     

林下参内生生防细菌的分离鉴定及定殖能力

【背景】多年生林下参在自然环境下生长多年,其体内存在的内生菌具有更强的适应性和定殖性,可以提高植物自身抗性,抑制病原菌的生长,更好地发挥与植物的互作。【目的】筛选定殖能力强、繁殖能力快且对病原菌具有拮抗作用的优势菌株。【方法】采用常规组织分离方法,从健康林下参根部组织中分离内生菌,通过对峙试验筛选出对人参病原菌有拮抗作用的内生细菌并对其以传统的鉴定方法进行鉴定。【结果】在得到的6株内生细菌中,菌株LXS-N2对人参立枯病病原菌、人参猝倒病病原菌均有明显抑菌性,而且具有定殖性好、繁殖快的特点,通过破坏病原真菌细胞壁和细胞膜以及改变菌丝形态从而抑制病原真菌生长。【结论】经形态学观察、生理生化反应及16S rRNA基因序列分析鉴定内生菌LXS-N2为贝莱斯芽孢杆菌,具有良好的应用开发潜力。     

辣椒内生细菌BS-1和BS-2在植物体内的定殖及鉴定

以抗利福平为标记,用浸种、涂叶和灌根方法接种,测定菌株在植物体内的定殖。结果表明,来自辣椒体内的BS_2和BS_1菌株不仅可在辣椒体内定殖,也可在番茄、茄子、黄瓜、甜瓜、西瓜、丝瓜、小白菜等植物体内定殖,BS_2菌株还可在水稻、小麦及豇豆等植物体内定殖,BS_2菌株的内生定殖宿主范围比BS_1菌株的广;另外BS_2菌株可在辣椒和白菜体内较长期定殖。用常规方法、Biolog及16S rRNA序列比较,两菌株鉴定为枯草芽杆菌内生亚种（Bacillus subtilis subsp. endophyticus）。     

草假单胞菌HT1在蚕豆根和茎的定殖特性及对内生细菌多样性的影响

[背景] 关于蚕豆内生生防细菌的定殖规律及其对内生细菌微生物多样性的研究鲜有报道。[目的] 明确草假单胞菌HT1在蚕豆的定殖特性以及对根茎部内生细菌群落多样性的影响。[方法] 采用抗生素标记法测定菌株HT1的定殖特性,利用高通量测序技术分析该菌灌根处理与对照处理蚕豆根茎部内生细菌的群落多样性。[结果] 菌株HT1的定殖数量为根>茎>叶,呈先升后降的趋势,根部、茎部、叶部在第7天达到最大值,在第83天仍能检测到标记菌株。灌根处理降低了内生细菌的丰富度,提高了根内生细菌物种多样性;灌根处理组根部厚壁菌门、放线菌门、拟杆菌门丰度显著提高,茎部变形菌门的相对丰度有所升高但无显著影响;在属水平上,灌根处理后,根部如Romboutsia、Mitsuaria、乳杆菌属、德沃斯氏菌属等属的相对丰度明显升高,茎部假单胞菌、Muribaculaceae、Elstera、鞘氨醇单胞菌属等属的相对丰度明显升高。[结论] HT1菌株能在蚕豆植株体内定殖达83 d以上,并且可以影响蚕豆植株内部的微生物群落结构组成,提高相关微生物的相对丰度。     

植物内生细菌的研究

植物内生细菌是能够定殖在植物细胞间隙或细胞内,并与寄主植物建立和谐联合关系的一类微生物I’］。自本世纪初开始,人们不断地从植物的根、叶、甚至茎和种子上分离并鉴定出多种微生物,但其在寄主植物中的生物学作用却未能引起研究者的重视。近年来,由于一些研究结果表明内生细菌能够作为外源基因的载体,又具有植物保护剂的功能,可以产生植物促生物质或可作为联合固氮菌剂,由此有关内生细菌的研究才引起植物学家和微生物学家的兴趣。l植物内生细菌的研究现状有关植物内生细菌的研究主要涉及两方面的内容：（l）生态学研究：内生细菌…     

Evaluation of endophytic colonization of Citrus sinensis and Catharanthus roseus seedlings by endophytic bacteria

Over the last few years, the endophytic bacterial community associated with citrus has been studied as an important component interacting with Xylella fastidiosa, the causal agent of citrus variegated chlorosis (CVC). This bacterium may also colonize some model plants, such as Catharanthus roseus and Nicotiana clevelandii. In the present study, we compared the endophytic colonization of Citrus sinensis and Catharanthus roseus using the endophytic bacteria Klebsiella pneumoniae. We chose an appropriate strain, K. pneumoniae 342 (Kp342), labeled with the GFP gene. This strain was inoculated onto seedlings of C. sinensis and C. roseus. The isolation frequency was determined one week after the inoculation and the endophytic colonization of K. pneumoniae was observed using fluorescence microscopy. Although the endophytic bacterium was more frequently isolated from C. roseus than from C. sinensis, the colonization profiles for both host plants were similar, suggesting that C. roseus could be used as a model plant to study the interaction between endophytic bacteria and X. fastidiosa.     

Endophytic bacteria of the rock-dwelling cactus Mammillaria fraileana affect plant growth and mobilization of elements from rocks

Mammillaria fraileana is a major pioneer, small cactus that harbors endophytic bacteria that have plant growth-promoting traits, including rock-weathering capacity. Our working hypothesis was that this functional group of endophytic bacteria assists in establishing pioneer plants on rocks. When these endophytic bacteria were inoculated on seedlings grown in rock substrate, mobilization of elements from the substrate increased at variable levels across combinations of substrates and inoculants. In plants grown in the rhyodacite substrate, where these cacti naturally grow, increased mobilization occurred in plants inoculated with several strains. Promotion of plant growth, manifested as an increase in dry weight, was greater in cacti inoculated with Enterobacter sakazakii M2PFe. Accumulation of nocturnal acids, indicating photosynthesis by crassulacean acid metabolism, was superior in plants inoculated with the endophytes Azotobacter vinelandii M2Per and Pseudomonas putida M5TSA. Inoculation with endophytes can stimulate plant growth of M. fraileana by mobilizing elements from rock, which can lead to higher photosynthetic activity and accumulation of biomass. Inoculation with P. putida M5TSA also led to accumulation of more total nitrogen than plants inoculated with a control nitrogen-fixing bacteria. Evidence of endophytic colonization is provided after initial inoculation of seedlings and re-isolation and sequencing of 16S DNA of recovered bacteria from developing disinfected plants. The associative interaction between pioneer cacti and their bacterial endophytes enable the host plants to grow in places where plants do not normally grow. Through colonization and establishment of pioneer plants, soil is created, which facilitates colonization by other desert species and contributes to the diversity of dry lands.     

Colonization behavior of bacterium Burkholderia cepacia inside the Oryza sativa roots visualized using green fluorescent protein reporter

Colonization behavior of endophytic bacteria Burkholderia cepacia strains RRE-3 and RRE-5 was studied in the seedlings of rice variety NDR97 using confocal laser scanning microscopy under controlled laboratory and greenhouse conditions. For studying colonization pattern, bacterial strains were tagged with pHRGFPGUS plasmid. The role of bacterial strains (both gfp/gus-tagged and untagged) in growth promotion was also studied. After coming into contact with the host root system the bacteria showed an irregular spreading. Dense colonization was observed on the primary and secondary roots and also on the junction of emergence of the lateral roots. Results showed that the colonization pattern of Burkholderia cepacia strains was similar to that of other endophytic bacteria isolated from non-legumes. Burkholderia cepacia got entry inside the root at the sites of emergence of lateral roots, without formation of infection threads as in the case of symbiotic rhizobacteria. Observations suggested that the endophytic bacterial strains RRE-3 and RRE-5 entered inside the rice roots in a progressive manner. Bacteria were found to line up along the intercellular spaces of adjoining epidermal cells adjacent to the lateral root junction, indicating endophytic colonization pattern of Burkholderia cepacia strains. Experiments with the rice seedlings inoculated with RRE-3 and RRE-5 strains revealed that both strains enhanced plant growth considerably when observed under laboratory and greenhouse conditions and produced significantly higher plant biomass. No considerable difference was observed between the gfp/gus-tagged and non-gfp/gus-tagged strains in the plant growth experiments both in the laboratory and greenhouse conditions.     

Endophytic bacteria as biocontrol agents against plant pathogens: current state-of-the-art

Most plants co-exist with fungal and bacterial endophytes. Generally, endophytes are beneficial microorganisms that colonize the internal tissues of their host plants. Plants derive several advantages from endophytic colonization, such as the biocontrol of phytopathogens and growth-promoting factors. In this review, we discuss the current knowledge of endophytic bacteria and their potential as natural biocontrol agents for plants.     

Masking of antibiotic-resistance upon recovery of endophytic bacteria

During studies on internal plant colonization by rhizosphere bacteria and endophytic bacteria over several years, we frequently observed lack of growth of rifampicin-resistant mutants (rif+) on tryptic soy agar amended with rifampicin (RTSA). Following seed treatment of cucumber with 6 species of rif+ rhizosphere bacteria in one experiment, all strains were recoverable on RTSA when external root colonization was monitored. Following trituration of surface-disinfested roots, only one strain grew directly on RTSA; however colonies isolated on tryptic soy agar (TSA) grew within 18 h after transfer to RTSA. We term this temporary loss of the antibiotic-resistant phenotype ‘antibiotic masking’. Antibiotic masking was also observed with isolation of 7 rif+ endophytic bacterial strains from inside stems of cotton and with isolation of mutants of bacterial endophytes resistant to polymyxin B sulfate from cotton plants. Rifampicin-masking was not accounted for in vitro by inhibitory compounds from cotton plant extracts, by bacterial growth on low nutrient agar, or by competition with other bacteria. Collectively, these results suggest that expression of antibiotic-resistance may be altered in planta, although causes for this antibiotic-masking remain to be elucidated, methods for quantifying internal plant colonization by rif+ bacteria should account for this possibility. ei]Section editor: R O D Dixon     

Effects of colonization of a bacterial endophyte,Azospirillum sp. B510, on disease resistance in tomato

A plant growth-promoting bacteria, Azospirillum sp. B510, isolated from rice, can enhance growth and yield and induce disease resistance against various types of diseases in rice. Because little is known about the interaction between other plant species and this strain, we have investigated the effect of its colonization on disease resistance in tomato plants. Treatment with this strain by soil-drenching method established endophytic colonization in root tissues in tomato plant. The endophytic colonization with this strain-induced disease resistance in tomato plant against bacterial leaf spot caused by Pseudomonas syringae pv. tomato and gray mold caused by Botrytis cinerea. In Azospirillum-treated plants, neither the accumulation of SA nor the expression of defense-related genes was observed. These indicate that endophytic colonization with Azospirillum sp. B510 is able to activate the innate immune system also in tomato, which does not seem to be systemic acquired resistance.     

Kinetics and strain specificity of rhizosphere and endophytic colonization by enteric bacteria on seedlings of Medicago sativa and Medicago truncatula

The presence of human-pathogenic, enteric bacteria on the surface and in the interior of raw produce is a significant health concern. Several aspects of the biology of the interaction between these bacteria and alfalfa (Medicago sativa) seedlings are addressed here. A collection of enteric bacteria associated with alfalfa sprout contaminations, along with Escherichia coli K-12, Salmonella enterica serotype Typhimurium strain ATCC 14028, and an endophyte of maize, Klebsiella pneumoniae 342, were labeled with green fluorescent protein, and their abilities to colonize the rhizosphere and the interior of the plant were compared. These strains differed widely in their endophytic colonization abilities, with K. pneumoniae 342 and E. coli K-12 being the best and worst colonizers, respectively. The abilities of the pathogens were between those of K. pneumoniae 342 and E. coli K-12. All Salmonella bacteria colonized the interiors of the seedlings in high numbers with an inoculum of 10(2) CFU, although infection characteristics were different for each strain. For most strains, a strong correlation between endophytic colonization and rhizosphere colonization was observed. These results show significant strain specificity for plant entry by these strains. Significant colonization of lateral root cracks was observed, suggesting that this may be the site of entry into the plant for these bacteria. At low inoculum levels, a symbiosis mutant of Medicago truncatula, dmi1, was colonized in higher numbers on the rhizosphere and in the interior by a Salmonella endophyte than was the wild-type host. Endophytic entry of M. truncatula appears to occur by a mechanism independent of the symbiotic infections by Sinorhizobium meliloti or mycorrhizal fungi.